The goal of this program is to characterize genes, and the pathways in which they participate, that are critical to the ionizing radiation resistance of Deinococcus radiodurans. This bacterium is the most radioresistant organism known. Furthermore, its repair of extensive DNA damage of almost all types is error-proof, producing no mutations. During the last project period we cloned seven genes important to the radioresistance of D. radiodurans. Three of these genes, uvsA, uvsB, and rec30, encode pivotal functions, since D. radiodurans strains mutant in any one of these genes are exquisitely sensitive to ionizing radiation. Complementation of a sensitive mutant by the corresponding cloned gene increases survival at 500,000 rads (5 kGy) from -10-15 to 100%, an enormous biological effect. Characterization of these seven genes, and others to be cloned, is a major goal for the coming project period. Of several approaches to this characterization, one important objective is to map tolerance and repair pathways by the deliberate construction of double, triple, and higher order mutants in genes implicated in radioresistance. We are able to do this because we have found that cloned genes are readily employed for targeted insertional mutagenesis in D. radiodurans. By assessment of sensitivity and repair properties of these multiple mutants we will create a map of reasonably characterized repair and tolerance pathways. Prior claims by others that cloned D. radiodurans DNA fragments in E coli encode radioresistance, have been withdrawn (see Background, Section III). This is not surprising, as we now know that deinococcal promoting sequences are ineffective in E coli. As another aspect of characterizing genes we have cloned, they will be introduced (in expression vectors) to E coli and its repair mutants. Cross complementation with E coli may provide important clues to the function of D. radiodurans genes. These studies will delineate the genetics of radioresistance in D. radiodurans, a necessary prerequisite to understanding its enzymology. Insight into its survival strategies may have broad implications for our understanding of radioresistance.